Electrofusion, that is, cell fusion in an electric field, has become of great interest as a powerful technique for the fusion of plant, animal, and microbial cells as it is simpler, more rapid, and less harmful to the cells than the conventional polyethylene glycol peg method and is available for mass production of fusion. Isolation and fusion of protoplasts from the phytopathogenic fungus. Even though transfer of a single gene from one plant to another is desirable and protoplast fusion facilitates easy monitoring of cell genetic changes. Top 15 applications of protoplast culture plant tissue. Protoplast fusion is important in biochemical andgenetic engineering studies, butmanydetails regarding its mechanismare notknown. It may allow the exchange of dna between unrelated. The uptake of isolated metaphase chromosomes has proven successful in plant protoplast. Plant protoplast fusion by electro cell fusion protocol cell type. This chapter discusses the plant protoplast fusion and somatic hybridization. Protoplast fusion technology was employed to reconstruct fusants from this fungus.
Identification and selection of somatic hybrid cells hybrid identification based on difference between the parental cells and hybrid cell with respect to pigmentation cytoplasmic markers fluorochromes like fitc fluoroscein isothiocyanate and ritc rhodamine isothiocyanate are used. Protoplasts can be isolated from a range of plant tissues. Among the many uses for these naked cells is transient or permanent genetic transformation by introduction of transgene dna, somatic hybridization by protoplast fusion of. It is the living matter enclosed by a plant cell membrane. Somatic hybrid protoplast fusion somatic cell hybridization plant protoplast leaf protoplast. The main objective of protoplast culture lies in the possibilities of fusion of one protoplast with another. Protoplast fusion technology and its biotechnological application.
Protoplast fusion allows the creation ofhybrid andcybrid. Establishment of method for protoplast fusion with pegmediated between jatropha curcas l. Which plants have already been bred with the help of protoplast fusion. Protoplast fusion has been used as a method to create mushroom hybrids especially when conventional method cannot be achieved. Plant protoplasts covers the techniques involved with, and uses of, protoplast technology. Protoplasts are isolated by two methods, mechanical and enzymatic. How to recognize seeds made with protoplast fusion until now organic plant breeders dont use protoplast fusion and also the organic seed producers know. In one study, a fusion rate of 5 to 12% was reported between protoplasts of c.
Fusion of protoplasts of potato and tomato, and production of hybrid plant pomato. The importance of protoplast sources and embedding systems is discussed. Plant protoplast fusion and growth of intergeneric hybrid. Progress in plant protoplast research springerlink. Membranefluidity may affect the rate ofprotoplast fusion. Fusion of proplast incudes three stages 1 agglutination 2 membrane fusion 3rounding off of the fused protoplast carlson et al 1972 first time fused the leaf protoplasts of nicotina gluca and n. For protoplast fusion it is important that the cell wall of plant and microorganisms is. Strain improvement of pleurotus species by protoplast fusion. Protoplasts are obtained from two different cells to fuse and form a hybrid protoplast which is called protoplast fusion. In other words protoplast fusion which is also called as somatic fusion is a type of of genetic modification in plants by which two different species of plants are fused together and a new hybrid plant is formed with the characteristics of both. The isolation, culture and fusion of protoplasts is a fascinating field in plant research.
The term protoplast was introduced by hanstein in 1880. Regeneration of new species and improved culture techniques opened new horizons for practical breeding in a number of crops. Simplynotes protoplast technologyprotoplast, protoplast. Protoplasts are plant cells which have lost their cell wall. Those fusion experiments involving petunia where somatic hybrid plants were successfully regenerated are discussed in sect. Agglutination and fusion of plant protoplasts by polyethylene glycol. The book discusses isolation, fusion, and culture of higher plant protoplasts, lower plant protoplasts, and bluegreen algal protoplast. Cells cultured at a low temperature contained larger proportions. Protoplasts regeneration and isolation plant tissue culture. Protoplasts are used in a number of ways for research and for plant improvement. Isolation of plant protoplasts by enzymatic method.
Efficient intergeneric fusion of pea pisum sativum l. The experimental conditions for plasmid transfer and genetic recombination in staphylococcus aureus and some coagulasenegative staphylococci by protoplast fusion are described. Protoplast fusion may be used to produce interspecific or even intergeneric hybrids. Indian journal of biotechnology vol 2, january 2003, pp 7684 protoplast fusion and brassica improvement pbkirtil, sprakash,srbhat2 andvlchopra 1department of plant sciences, university of hyderabad, hyderabad 500046 2national research centre on plant biotechnology, indian agricultural research institute. Read this article to learn about the isolation of plant protoplasts by enzymatic method, plant protoplast culture, somatic hybridization and fusion techniques used in plant protoplasts.
Plant protoplast fusion by electrocellfusion and by. Protoplast fusion article about protoplast fusion by the. Potentials of protoplast fusion in plant breeding programmes. Introduction a protoplast is a plant, bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means. Pdf protoplast fusion technology and its biotechnological. Establishment of method for protoplast fusion with peg. General plant protoplast and celery apium graveolens l. Isolation and callus production from cotyledon protoplasts. Protoplast fusion becomes an important tool of gene manipulation because it breakdown the barriers to genetic exchange imposed by conventional mating systems. The purpose of this section is to relate and summarize the observations of those protoplast fusion experiments in which one partner was a petunia species and hybrid cells or their derived calli were produced, but hybrid plants were not regenerated table 11. Somatic hybrids between a herbaceous and two tree datura species. A mixture of carbohydrases can be used to degrade plant tissue, producing a protoplast suspension which can easily be seen under a microscope.
Isolation and fusion of protoplasts from mesophyll cells of dendrobium pompadour kamnoon kanchanapooma, saowarath jantarob and duangkae rakchadb a department of biology, faculty of science, prince of songkla university, hatyai, songkhla 90112, thailand. That is, it contains all the functional part of the cell without the barrier of the cell wall. Plasmid transfer and genetic recombination by protoplast. Have their cell wall only partially removed more generally protoplast refers. If so, certain kinds of protoplast fusion would be legally allowed in organic farming. And brachiaria decumbens by zainah binti daud thesis submitted in fulfillment of the requirements for the degree of doctor of philosophy september 2015. Our representative from the ministry of agriculture in the scof is trying to get an answer to this question. Protoplast fusion facilitates mixing of two genomes and could be exploited in crosses which are not possible by conventional techniques due to incompatibility. This chapter discusses plant protoplast fusion by electromanipulation. Plant protoplast fusion by electromanipulation sciencedirect. Cocking plant protoplast fusion somatic hybridization of plants relies on the availability of efficient and reproducible methods for the induced. Protoplast isolation and culture of dendrobium sonia bom 17.
Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0. Protoplast fusion induced by the application of electric shock 18. Wepreviously proposed that membranefluidity is determined bythe phase transition ofthe phospholipid molecularspecies 3, 17. Plant protoplasts a biotechnological tool for plant improvement. Protoplast isolation and culture plant tissue culture. Impacts of banning protoplast fusion on the range of. Two glass capillary microelectrode are placed in contact with the protoplasts. Tissue slices weregently shaken, in the dark, at 30cfor 4h.
Agricultural applications of plant protoplast fusion nature. Protoplast isolation science and plants for schools. Have their cell wall entirely removed spheroplasts. Rapid progress occurred after 1980 in protoplast fusion to improve plant genetic material, and the development of transgenic plants. Theprotoplasts were passed through an 80,tm nylon screen and purified on an isoosmotic sucrose gradient as described by. Impacts of banning protoplast fusion, 2008 8 it must be noted that protoplast fusion is carried out on the basis of vegetative plant cells.
Identification and selection of somatic hybrid cells. Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the. Mertens and jacobsen, 1993 and cultured in hormone. Often the presence of polyethylene glycol or electric fields will enhance the frequency of fusion. Pdf protoplast and cell culture methods in somatic. Plant protoplast fusion and growth of intergeneric hybrid cells k. Protoplast technologyprotoplast, protoplast isolation. For school work, this procedure does not require an aseptic environment if the protoplasts are not to be. Whole plants can be regenerated from protoplasts grown on solid or liquid media. Protoplast fusion and growth of hybrid cells 219 table 6. Protoplasts regeneration and isolation ptc protoplasts are cells that have had their cell walls removed, and possess plasma membrane and all other remaining cellular components.
Protoplast fusion definition of protoplast fusion by. This procedure provides a valuable method for genetic information transfer and gene analysis. Protoplast isolation and culture of dendrobium sonia bom 17 yuphin khentry1, ampaiwan paradornuvat 1, sureeya tantiwiwat 2, salak phansiri3 and niphone thaveechai1 abstract the optimization of dendrobium sonia bom 17 protoplast isolation and culture conditions were investigated. It refers to the cellular content excluding cell wall or can also be called as naked plant cell. Isolation and fusion of protoplasts from mesophyll cells. Protoplast and cell culture methods in somatic hybridization in higher plants. This technique is known as electrofusion of protoplasts. Plant protoplast fusion may be performed by electro cell fusion using our ecfg21 device which has replaced the lf101 and lf201 devices download. Fusio the stated methodologies illustrate the use of our lf201 or lf101 devices now replaced by the ecfg21 device.
An electric field of low strength 10kv m3 give rise to dielectrophoretic pole generation within the protoplast suspension. Cocking department of genetics, university of leeds, leeds ls2 9jt, england, and plant genetic manipulation group, department of botany, university of nottingham, nottingham ng7 2rd, england abstract. Citrus asymmetric somatic hybrids produced via fusion of. This lecture explains about the protoplast fusion technique also known as somatic cell fusion which allows us to produce plant cell hybrids with. Protoplast fusion technique has a great potential for genetic analysis and for strain. Protoplast fusion technology vary protoplast from speciesgenera, fusion to create new genotypes, plant regeneration organogenesissomatic embryogenesis, isolated in large quantities, protoplasts must be. It began some 15 years ago, when gary harman, a cornell professor of horticultural science, and tom stasz, then a research associate, took two different strains of trichoderma that occur naturally in soil and, using protoplast fusion, bioengineered a new strain. Pdf protoplast fusion for phaseolus breeding researchgate. Procedure for successful somatic hybridization is as below.
The isolation and culture media used vary with the species and with the tissue from which the protoplasts were isolated. Gamborg prairie regional laboratory, national research council of canada, saskatoon, saskatchewan s7n ow9, canada. Protoplast fusion would continue to be an existing area of research in modern biotechnology 11. Protoplast fusion technology for somatic hybridisation in. Protoplasts are cells of plants, fungi, or bacteria in which the cell wall has been removed, but the plasma membrane is intact. Interspecific and intergeneric fusions of plant protoplasts were induced by. A robotic platform for highthroughput protoplast isolation and transformation elizabeth m. Somatic hybridization involves four discrete, yet interrelated, stages. Mechanical isolation method of protoplasts is no more practically used. Therefore this technology can not directly be assigned to the field of genetic engineering where the new combination of genetic material is performed at dna level.
Protoplast fusion technology and its biotechnological. Production of somatic hybrid plants through protoplast. Some of the advantages of protoplast fusion are large scale reproducible isolation of protoplasts from various tissues and more or less universal application. Plant protoplast fusion and growth of intergeneric hybrid cells. The relation between the composition of the phospholipid molecular species in a cell membrane and the velocity of protoplast fusion was studied using cells cultured at a low temperature 10 c. Nootjaree tudses1, siripong premjet,2,3 and duangporn premjet1,3. Lf101 electro cell fusion generator electrodechamber. Electro fusion recently, mild electrical stimulation is being used to fuse protoplasts.
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